© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1628-1638 doi:10.1242/dev.111922
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Lineage tracing of neuromesodermal progenitors reveals novel Wnt-dependent roles in trunk progenitor cell maintenance and differentiation
ABSTRACT In the development of the vertebrate body plan, Wnt3a is thought to promote the formation of paraxial mesodermal progenitors (PMPs) of the trunk region while suppressing neural specification. Recent lineage-tracing experiments have demonstrated that these trunk neural progenitors and PMPs derive from a common multipotent progenitor called the neuromesodermal progenitor (NMP). NMPs are known to reside in the anterior primitive streak (PS) region; however, the extent to which NMPs populate the PS and contribute to the vertebrate body plan, and the precise role that Wnt3a plays in regulating NMP self-renewal and differentiation are unclear. To address this, we used cell-specific markers (Sox2 and T) and tamoxifen-induced Cre recombinase-based lineage tracing to locate putative NMPs in vivo. We provide functional evidence for NMP location primarily in the epithelial PS, and to a lesser degree in the ingressed PS. Lineage-tracing studies in Wnt3a/β-catenin signaling pathway mutants provide genetic evidence that trunk progenitors normally fated to enter the mesodermal germ layer can be redirected towards the neural lineage. These data, combined with previous PS lineage-tracing studies, support a model that epithelial anterior PS cells are Sox2+T+ multipotent NMPs and form the bulk of neural progenitors and PMPs of the posterior trunk region. Finally, we find that Wnt3a/β-catenin signaling directs trunk progenitors towards PMP fates; however, our data also suggest that Wnt3a positively supports a progenitor state for both mesodermal and neural progenitors. KEY WORDS: Neural progenitor, Neuromesodermal progenitor, Wnt signaling, Brachyury, Paraxial mesoderm, Sox2
INTRODUCTION
How progenitor populations are directed to differentiate into different cell lineages is poorly understood in vivo, particularly for the embryonic progenitors of the neural and mesodermal lineages. Neuroectoderm and mesoderm progenitors were originally thought to arise as separate descendants of the pluripotent epiblast during early gastrulation (Bellairs, 1986; Garcia-Martinez and Schoenwolf, 1992; Tam and Behringer, 1997). However, in mammalian embryos, a common neuromesodermal progenitor (NMP) is thought to give rise to both neural and paraxial mesoderm progenitors (PMPs) (Cambray and Wilson, 2002, 2007; Tam and Beddington, 1987; Tzouanacou et al., 2009; Wilson et al., 2009). The descendants of the neural Center for Cancer Research, Cancer and Developmental Biology Laboratory, Cell Signaling in Vertebrate Development Section, NCI-Frederick, NIH, Frederick, MD 21702, USA. *Author for correspondence ([email protected]) Received 25 April 2014; Accepted 11 March 2015
1628
progenitors give rise to the spinal cord, whereas the PMPs can differentiate into bone, cartilage, muscle, dermis and connective tissue. Thus, the potential of NMPs is remarkable, perhaps accounting for the majority of tissues that form the trunk and tail. Multipotent progenitors have been identified in the mouse primitive streak (PS) region near the embryonic node by cell-tracing experiments showing that individual or groups of progenitors give rise to both neural and paraxial mesoderm (PM) (Cambray and Wilson, 2002, 2007; Tzouanacou et al., 2009; Wilson et al., 2009). Regional PS progenitors show the capacity to contribute to multiple cell types along the body axis and can be passaged through multiple successive host embryos, suggesting that the PS region contains stem cell populations (Cambray and Wilson, 2002; Wilson et al., 2009). However, recognizing axial stem cells in vivo by molecular criteria remains a challenge as the PS region is a site of dynamic and continuous cell movement with different, indistinguishable populations of cells entering, exiting and residing in the PS (GarciaMartinez and Schoenwolf, 1992; Schoenwolf et al., 1992; Wilson and Beddington, 1996). NMPs are proposed to co-express Sox2, a pluripotency and neural progenitor marker, and the PS marker T, also known as brachyury (Martin and Kimelman, 2012; Wilson et al., 2009). Although Sox2/T co-expressing regions have been documented in the PS region in mammals (Tsakiridis et al., 2014), no study has demonstrated that Sox2/T co-expression functionally represents multipotent NMPs. One way to address whether T-expressing cells include NMPs is to take a gene-specific transgenic lineage-tracing approach. The recently developed TCreERT2 transgenic line, in which the tamoxifen (TAM)-inducible CreERT2 recombinase is driven by the T promoter, has the potential to trace NMPs in vivo as it is expressed in the anterior epithelial PS region where NMPs are thought to exist (Anderson et al., 2013; Wilson et al., 2009). TCreERT2 also has the advantage of excluding pluripotent epiblast stem cells that are capable of giving rise to most embryonic cell types, including neural and mesodermal cells. Previous transgenic tracing experiments have successfully used TAM-inducible Cre-based transgenics to follow the population dynamics of diverse progenitor populations in vivo (Boyle et al., 2008; Göthert et al., 2005; Masahira et al., 2006; Schepers et al., 2012; Srinivasan et al., 2007). Wnt3a has been proposed to be a crucial regulator of NMP maintenance and differentiation, and presumably does so through β-catenin/Tcf transcriptional complexes and the subsequent activation of downstream target genes (Clevers and Nusse, 2012) such as T (Yamaguchi et al., 1999). In the absence of Wnt3a or T, the embryo fails to generate the posterior-most ∼55 of 65 skeletal elements/vertebrae, resulting in a severe posterior axis truncation (Herrmann, 1992; Takada et al., 1994). The loss of
DEVELOPMENT
Robert J. Garriock, Ravindra B. Chalamalasetty, Mark W. Kennedy, Lauren C. Canizales, Mark Lewandoski and Terry P. Yamaguchi*
skeletal elements corresponds to a reduction in progenitor markers, indicating a collapse in the progenitor populations that build the embryonic trunk (Yamaguchi et al., 1999). Importantly, Wnt3a, Tcf1;Lef1 double mutants, and T mutant embryos show an expansion of neural tissue in the form of an ectopic neural tube, giving support to the hypothesis that the Wnt3a/β-catenin/Tcf1Lef1/T axis is directly regulating the differentiation of NMPs into neural or mesodermal progenitors (Galceran et al., 1999; Herrmann, 1992; Yamaguchi et al., 1999; Yoshikawa et al., 1997). Single cell studies in zebrafish further this argument by showing that embryonic progenitors will selectively form striated skeletal muscle tissue when exposed to high Wnt signaling and, by contrast, form neural tissue when Wnt signaling is inhibited (Martin and Kimelman, 2012). From these, and other studies, a model of Wnt3a function has evolved to incorporate the concept of the NMP (Fig. 1A) (Galceran et al., 1999; Li and Storey, 2011; Martin and Kimelman, 2008, 2012; Takada et al., 1994; Yamaguchi et al., 1999; Yoshikawa et al., 1997). This model predicts that Wnt has a direct role in NMP maintenance, inducing PM cell fate and repressing neural cell fate. However, the model remains hypothetical and has not been directly tested in the native mammalian niche. The Wnt3a-null and conditional Ctnnb1 (β-catenin) mouse mutants provide excellent opportunities to
Development (2015) 142, 1628-1638 doi:10.1242/dev.111922
study NMP behavior in vivo as the fate of these cells can be dramatically modulated through these single gene mutations. Here, we show that Wnt3a is required for regulating the balance of PM and neural tissues through the regulation of progenitor populations located at the posterior pole of the extending anteriorposterior axis. We further show that Wnt3a/β-catenin signals play a key role in maintaining Sox2+T+ NMPs and, unexpectedly, do not repress neural fates. RESULTS Imbalance of neural progenitors and PMPs, and differentiated descendants in Wnt3a−/− mutants
To assess neural progenitors and PMPs in Wnt3a−/− mutants, we examined representative markers of each population in sections taken just posterior to the forelimb, corresponding to the level of the 13th-16th somite (s13-16) of wild-type E9.5 embryos. Phenotypic differences are clearly evident between control and Wnt3a−/− mutant embryos at this stage and axial level (Takada et al., 1994; Yoshikawa et al., 1997). E9.5 Wnt3a−/− mutant embryos showed enlarged and malformed neural tissue evident by detection of the neural progenitor marker Sox2, while fibronectin expression revealed a reduced PM (Fig. 1B). Quantification showed that Wnt3a−/− mutants had significantly more neural progenitors and fewer Fig. 1. Loss of PMPs and expansion of neural progenitors in Wnt3a−/− mutants is not due to changes in cell proliferation. (A) Proposed model of Wnt3a function in NMPs. Wnt3a maintains NMPs and promotes PM differentiation while inhibiting neural differentiation. (B) Detection of Sox2 (neural progenitors) and fibronectin (Fn1) (mesodermal mesenchyme) in the s13-16 region of E9.5 control and Wnt3a−/− embryos. (C) Graph of total cell counts of neural and mesodermal cells at E9.5 (n=4 control, n=5 Wnt3a−/−). Data are mean±s.e.m. P
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Lineage tracing of neuromesodermal progenitors reveals novel Wnt-dependent roles in trunk progenitor cell maintenance and differentiation
ABSTRACT In the development of the vertebrate body plan, Wnt3a is thought to promote the formation of paraxial mesodermal progenitors (PMPs) of the trunk region while suppressing neural specification. Recent lineage-tracing experiments have demonstrated that these trunk neural progenitors and PMPs derive from a common multipotent progenitor called the neuromesodermal progenitor (NMP). NMPs are known to reside in the anterior primitive streak (PS) region; however, the extent to which NMPs populate the PS and contribute to the vertebrate body plan, and the precise role that Wnt3a plays in regulating NMP self-renewal and differentiation are unclear. To address this, we used cell-specific markers (Sox2 and T) and tamoxifen-induced Cre recombinase-based lineage tracing to locate putative NMPs in vivo. We provide functional evidence for NMP location primarily in the epithelial PS, and to a lesser degree in the ingressed PS. Lineage-tracing studies in Wnt3a/β-catenin signaling pathway mutants provide genetic evidence that trunk progenitors normally fated to enter the mesodermal germ layer can be redirected towards the neural lineage. These data, combined with previous PS lineage-tracing studies, support a model that epithelial anterior PS cells are Sox2+T+ multipotent NMPs and form the bulk of neural progenitors and PMPs of the posterior trunk region. Finally, we find that Wnt3a/β-catenin signaling directs trunk progenitors towards PMP fates; however, our data also suggest that Wnt3a positively supports a progenitor state for both mesodermal and neural progenitors. KEY WORDS: Neural progenitor, Neuromesodermal progenitor, Wnt signaling, Brachyury, Paraxial mesoderm, Sox2
INTRODUCTION
How progenitor populations are directed to differentiate into different cell lineages is poorly understood in vivo, particularly for the embryonic progenitors of the neural and mesodermal lineages. Neuroectoderm and mesoderm progenitors were originally thought to arise as separate descendants of the pluripotent epiblast during early gastrulation (Bellairs, 1986; Garcia-Martinez and Schoenwolf, 1992; Tam and Behringer, 1997). However, in mammalian embryos, a common neuromesodermal progenitor (NMP) is thought to give rise to both neural and paraxial mesoderm progenitors (PMPs) (Cambray and Wilson, 2002, 2007; Tam and Beddington, 1987; Tzouanacou et al., 2009; Wilson et al., 2009). The descendants of the neural Center for Cancer Research, Cancer and Developmental Biology Laboratory, Cell Signaling in Vertebrate Development Section, NCI-Frederick, NIH, Frederick, MD 21702, USA. *Author for correspondence ([email protected]) Received 25 April 2014; Accepted 11 March 2015
1628
progenitors give rise to the spinal cord, whereas the PMPs can differentiate into bone, cartilage, muscle, dermis and connective tissue. Thus, the potential of NMPs is remarkable, perhaps accounting for the majority of tissues that form the trunk and tail. Multipotent progenitors have been identified in the mouse primitive streak (PS) region near the embryonic node by cell-tracing experiments showing that individual or groups of progenitors give rise to both neural and paraxial mesoderm (PM) (Cambray and Wilson, 2002, 2007; Tzouanacou et al., 2009; Wilson et al., 2009). Regional PS progenitors show the capacity to contribute to multiple cell types along the body axis and can be passaged through multiple successive host embryos, suggesting that the PS region contains stem cell populations (Cambray and Wilson, 2002; Wilson et al., 2009). However, recognizing axial stem cells in vivo by molecular criteria remains a challenge as the PS region is a site of dynamic and continuous cell movement with different, indistinguishable populations of cells entering, exiting and residing in the PS (GarciaMartinez and Schoenwolf, 1992; Schoenwolf et al., 1992; Wilson and Beddington, 1996). NMPs are proposed to co-express Sox2, a pluripotency and neural progenitor marker, and the PS marker T, also known as brachyury (Martin and Kimelman, 2012; Wilson et al., 2009). Although Sox2/T co-expressing regions have been documented in the PS region in mammals (Tsakiridis et al., 2014), no study has demonstrated that Sox2/T co-expression functionally represents multipotent NMPs. One way to address whether T-expressing cells include NMPs is to take a gene-specific transgenic lineage-tracing approach. The recently developed TCreERT2 transgenic line, in which the tamoxifen (TAM)-inducible CreERT2 recombinase is driven by the T promoter, has the potential to trace NMPs in vivo as it is expressed in the anterior epithelial PS region where NMPs are thought to exist (Anderson et al., 2013; Wilson et al., 2009). TCreERT2 also has the advantage of excluding pluripotent epiblast stem cells that are capable of giving rise to most embryonic cell types, including neural and mesodermal cells. Previous transgenic tracing experiments have successfully used TAM-inducible Cre-based transgenics to follow the population dynamics of diverse progenitor populations in vivo (Boyle et al., 2008; Göthert et al., 2005; Masahira et al., 2006; Schepers et al., 2012; Srinivasan et al., 2007). Wnt3a has been proposed to be a crucial regulator of NMP maintenance and differentiation, and presumably does so through β-catenin/Tcf transcriptional complexes and the subsequent activation of downstream target genes (Clevers and Nusse, 2012) such as T (Yamaguchi et al., 1999). In the absence of Wnt3a or T, the embryo fails to generate the posterior-most ∼55 of 65 skeletal elements/vertebrae, resulting in a severe posterior axis truncation (Herrmann, 1992; Takada et al., 1994). The loss of
DEVELOPMENT
Robert J. Garriock, Ravindra B. Chalamalasetty, Mark W. Kennedy, Lauren C. Canizales, Mark Lewandoski and Terry P. Yamaguchi*
skeletal elements corresponds to a reduction in progenitor markers, indicating a collapse in the progenitor populations that build the embryonic trunk (Yamaguchi et al., 1999). Importantly, Wnt3a, Tcf1;Lef1 double mutants, and T mutant embryos show an expansion of neural tissue in the form of an ectopic neural tube, giving support to the hypothesis that the Wnt3a/β-catenin/Tcf1Lef1/T axis is directly regulating the differentiation of NMPs into neural or mesodermal progenitors (Galceran et al., 1999; Herrmann, 1992; Yamaguchi et al., 1999; Yoshikawa et al., 1997). Single cell studies in zebrafish further this argument by showing that embryonic progenitors will selectively form striated skeletal muscle tissue when exposed to high Wnt signaling and, by contrast, form neural tissue when Wnt signaling is inhibited (Martin and Kimelman, 2012). From these, and other studies, a model of Wnt3a function has evolved to incorporate the concept of the NMP (Fig. 1A) (Galceran et al., 1999; Li and Storey, 2011; Martin and Kimelman, 2008, 2012; Takada et al., 1994; Yamaguchi et al., 1999; Yoshikawa et al., 1997). This model predicts that Wnt has a direct role in NMP maintenance, inducing PM cell fate and repressing neural cell fate. However, the model remains hypothetical and has not been directly tested in the native mammalian niche. The Wnt3a-null and conditional Ctnnb1 (β-catenin) mouse mutants provide excellent opportunities to
Development (2015) 142, 1628-1638 doi:10.1242/dev.111922
study NMP behavior in vivo as the fate of these cells can be dramatically modulated through these single gene mutations. Here, we show that Wnt3a is required for regulating the balance of PM and neural tissues through the regulation of progenitor populations located at the posterior pole of the extending anteriorposterior axis. We further show that Wnt3a/β-catenin signals play a key role in maintaining Sox2+T+ NMPs and, unexpectedly, do not repress neural fates. RESULTS Imbalance of neural progenitors and PMPs, and differentiated descendants in Wnt3a−/− mutants
To assess neural progenitors and PMPs in Wnt3a−/− mutants, we examined representative markers of each population in sections taken just posterior to the forelimb, corresponding to the level of the 13th-16th somite (s13-16) of wild-type E9.5 embryos. Phenotypic differences are clearly evident between control and Wnt3a−/− mutant embryos at this stage and axial level (Takada et al., 1994; Yoshikawa et al., 1997). E9.5 Wnt3a−/− mutant embryos showed enlarged and malformed neural tissue evident by detection of the neural progenitor marker Sox2, while fibronectin expression revealed a reduced PM (Fig. 1B). Quantification showed that Wnt3a−/− mutants had significantly more neural progenitors and fewer Fig. 1. Loss of PMPs and expansion of neural progenitors in Wnt3a−/− mutants is not due to changes in cell proliferation. (A) Proposed model of Wnt3a function in NMPs. Wnt3a maintains NMPs and promotes PM differentiation while inhibiting neural differentiation. (B) Detection of Sox2 (neural progenitors) and fibronectin (Fn1) (mesodermal mesenchyme) in the s13-16 region of E9.5 control and Wnt3a−/− embryos. (C) Graph of total cell counts of neural and mesodermal cells at E9.5 (n=4 control, n=5 Wnt3a−/−). Data are mean±s.e.m. P
